The cDNA encoding epsilon-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant His6- tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His6 tag. Radiolabeled coatomer was employed to establish that all the subunits of the coatomer enter coated vesicles as an intact unit.